Interferon (IFNs) are a family of cytokines with diverse biological functions, including antitumor, immunomodulatory, antiviral and antiparasitic actions. At present, the IFN family includes more than 20 different proteins. Initially, IFN""s were classified by source as leucocyte, fibroblast, or immune IFNs. Initially, IFNs were also grouped into two types with Type 1 comprising fibroblast and leucocyte IFNs and type II comprising immune IFNs. Based on their genetic similarities and differences, the type I IFNs are currently divided into the following three classes: IFN-xcex1, IFN-xcex2, and IFN-xcfx89. The type II IFNs are classified as IFN-xcex3.
IFNs act by binding to specific cell receptors, which are found on the surface of most cells, and causing the translocation to the nucleus of cytoplasmic transcription factors that enhance or suppress the expression of specific genes. The products of these interferon-stimulated genes (hereinafter referred to as ISGs) are primarily polypeptides that act as mediators of the biological activities associated with the respective IFN. IFN-xcex1 and IFN-xcex2 bind to the same receptor and activate transcription of ISGs through the assembly and translocation from the cytoplasm to the nucleus of ISGF3, a multisubunit transcription factor that interacts with a cis-acting DNA regulatory element termed the interferon-stimulated response element or ISRE.
Transcription of the ISGs stimulated by IFN-xcex1 and IFN-xcex2 occurs in the absence of ongoing protein synthesis, and is therefore mediated directly by the interaction of either IFN-xcex1 or IFN-xcex2 with their common receptor.
IFN-xcex2 is a particularly useful cytokine which has been shown to be effective in decreasing the relapse rate, relapse severity, progression of neurological disability and development of new brain lesions, as observed with brain magnetic resonance imaging, in relapsing-remitting multiple sclerosis patients. Unfortunately, IFN-xcex2 can cause side effects in such patients. Moreover, a substantial number of the patients treated with IFN-xcex2 develop neutralizing antibodies. In addition, IFN-xcex2 must be administered by injection. There are also concerns that IFN-xcex2 may not penetrate certain tissue compartments. Accordingly, efforts are currently underway to identify other compounds, particularly small molecular weight molecules, that induce an IFN-xcex2 type response, hereinafter referred to as xe2x80x9cIFN-xcex2 mimicsxe2x80x9d. These efforts are directed at identifying IFN-xcex2 mimics that can be administered orally and that freely permeate tissues. The efforts are also directed at identifying IFN-xcex2 mimics that are less toxic than naturally-occurring or recombinant forms of IFN-xcex2.
In certain instances, such as autoimmune diseases, IFN-xcex1 or IFN-xcex2 can induce a deleterious inflammatory reaction in an individual. Accordingly efforts are also underway to identify compounds, hereinafter referred to as xe2x80x9cIFN-xcex2 inhibitorsxe2x80x9d, that block the IFN-xcex2 type components of this reaction.
The efforts to identify IFN-xcex2 mimics and IFN-xcex2 inhibitors have been hampered by the fact that there are no known ISG products whose synthesis is selectively induced by IFN-xcex2 as compared to IFN-xcex1 and thus can be used to give the full measure of an IFN-xcex2 type response. Accordingly, it is desirable to have an ISG product whose synthesis is selectively induced by IFN-xcex2 as compared to IFN-xcex1. It is also desirable to have a system that can be used to identify compounds that selectively induce an IFN-xcex2 type response as compared to an IFN-xcex1 type response. An in vitro system that can be used to screen IFN-xcex2 mimics and that can be used to assess the potency of the IFN-xcex2 mimics is especially desirable. It is also desirable to have a system that can be used to screen IFN-xcex2 inhibitors.
The present invention provides a system for screening IFN-xcex2 mimics or IFN-xcex2 inhibitors. The system comprises a polynucleotide that comprises a DNA sequence encoding a unique chemokine promoter, hereinafter referred to as the xe2x80x9cBeta R1 promoterxe2x80x9d, operatively linked to a reporter gene that encodes an assayable product.
The present invention also provides a method of employing the system to assay the concentration of IFN-xcex2 in a sample and to screen putative IFN-xcex2 mimics. In one embodiment, the method comprises the steps of providing cultured cells transfected with a polynucleotide construct that comprises a DNA sequence that encodes the Beta R1 promoter operatively linked to a reporter gene that encodes an assayable product; treating the transfected cells with the putative IFN-xcex2 mimic or the sample; detecting the assayable product produced by the treated cells. Synthesis of the assayable product by the cells treated with the putative IFN-xcex2 mimic or the sample is indicative of the IFN-xcex2 response. In another embodiment, the method comprises the steps of treating a group of cultured cells with the putative IFN-xcex2 mimic or sample and detecting the presence of the Beta R1 transcript in the treated cells; wherein the presence of the Beta R1 transcript in the treated cells is indicative of the IFN-xcex2 response.
The present invention also provides an isolated Beta R1 polypeptide, whose production is selectively induced by IFN-xcex2 as compared to IFN-xcex1. The Beta R1 polypeptide is an inducible CXC, non-ELR chemokine whose expression is stimulated in T cells, astrocytes, and fibroblasts that have been exposed to IFN-xcex2. The present invention also provides an isolated polynucleotide. that encodes the Beta R1 polypeptide.